Journal: Journal of Neuroinflammation

Article Title: Decreased progranulin levels in patients and rats with subarachnoid hemorrhage: a potential role in inhibiting inflammation by suppressing neutrophil recruitment

doi: 10.1186/s12974-015-0415-4

Figure Lengend Snippet: ELISA analysis of PGRN, MPO, IL-β, and TNF-α in the CSF of patients. The concentration of PGRN decreased after SAH and was lowest in the 1–3 days group ( a ). However, the concentration of MPO, IL-β, and TNF-α increased after SAH and was highest in the 1–3 days group ( b – d ). Data are expressed as the mean ± SEM from six rats. * p < 0.05 compared with the control group, ns p > 0.05 compared with the control group

Article Snippet: The inflammatory cytokine levels of brain tissue were quantified using enzyme-linked immunosorbent assay (ELISA) kits specific for rats according to the manufacturer’s instructions (PGRN, P28799, from Raybiotech, USA; MPO, ab119605, from Abcam, USA; tumor necrosis factor-α (TNF-α), 950.090.096 and 865.000.096, from Diaclone Research, France; IL-1β, 850.006.096 and 670.040.096, from Diaclone Research, France).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Control

Journal: Journal of Neuroinflammation

Article Title: Decreased progranulin levels in patients and rats with subarachnoid hemorrhage: a potential role in inhibiting inflammation by suppressing neutrophil recruitment

doi: 10.1186/s12974-015-0415-4

Figure Lengend Snippet: ELISA analysis of IL-β and TNF-α in the brain cortex of rats 24 h after SAH. The concentrations of IL-β ( a ) and TNF-α ( b ) significantly increased at 24 h after SAH and decreased after administration of r-PGRN. No difference was detected between the untreated SAH group and the PBS-treated SAH group. Data are expressed as the mean ± SEM from six rats. *** p < 0.001 compared with the sham group, ns p > 0.05 compared with the SAH group, ## p < 0.01 compared with the SAH group

Article Snippet: The inflammatory cytokine levels of brain tissue were quantified using enzyme-linked immunosorbent assay (ELISA) kits specific for rats according to the manufacturer’s instructions (PGRN, P28799, from Raybiotech, USA; MPO, ab119605, from Abcam, USA; tumor necrosis factor-α (TNF-α), 950.090.096 and 865.000.096, from Diaclone Research, France; IL-1β, 850.006.096 and 670.040.096, from Diaclone Research, France).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Glia

Article Title: iPSC-derived microglia carrying the TREM2 R47H/+ mutation are proinflammatory and promote synapse loss.

doi: 10.1002/glia.24485

Figure Lengend Snippet: FIGURE 1 The TREM2 R47H/+ mutation alters microglial gene expression and induces a proinflammatory signature. (a) Schematic of CRISPR mutagenesis and generation of TREM2 R47H/+ mutant microglia. (b) Diagram of TREM2 protein and downstream effectors. Location of the R47H/+ mutation is indicated. Chromatogram traces show successful editing of the R47H sequence in TREM2. (c) Quantification of TREM2 mRNA level in TREM2 R47H/+ and isogenic control microglia. n = 6 for each of CTRL-A, CTRL-B, R47H-A, and R47H-B. (d) Images and (e) quantification of western blots for different forms of TREM2. TREM2 null induced pluripotent stem cell microglia (see Section 2) were included to control for antibody specificity. Actin serves as loading control. n = 6 for each of CTRL-A, CTRL-B, R47H-A, and R47H-B. (f) Quantification of ELISA for soluble TREM2 levels in culture media from control and TREM2 R47H/+ microglia. n = 3 for each of CTRL-A, CTRL-B, R47H-A, R47H-B, and null. ***p < .001. Student's t-test. (g) Principal component analysis plot for control and TREM2 R47H/+ biological and technical replicates. (h) Volcano plot and (i) enriched GO terms for genes differentially expressed between control and TREM2 R47H/+ microglia. (j) Heatmap of the gene expression Z-score for genes in the “Inflammatory response” GO term in control and TREM2 mutant microglia that showed significantly increased expression in TREM2 R47H/+ microglia.

Article Snippet: Human IL-6 or TNF-α Quantkine ELISA Kits (R + D Systems) were used.

Techniques: Mutagenesis, Gene Expression, CRISPR, Sequencing, Control, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Glia

Article Title: iPSC-derived microglia carrying the TREM2 R47H/+ mutation are proinflammatory and promote synapse loss.

doi: 10.1002/glia.24485

Figure Lengend Snippet: FIGURE 2 TREM2 R47H/+ microglia show exaggerated responses to inflammatory stimuli. (a) Quantification of interleukin-6 (IL-6) enzyme- linked immunosorbent assays (ELISAs) from culture media following treatment with 100 ng/mL lipopolysaccharide (LPS) (left) or 50 ng/mL interferon-γ (IFN-γ) (right). n = 8 for CTRL-A, CTRL-B, R47H-A, and R47H-B for all baseline conditions; n = 9 for CTRL-A, CTRL-B, R47H-A, and R47H-B for all LPS and IFN-γ treated conditions. ***p < .001. One-way analysis of variance (ANOVA) with Sidak's test. (b) Quantification of tumor necrosis factor-α (TNF-α) ELISAs from culture media following treatment with 100 ng/mL LPS (left) or 50 ng/mL IFN-γ (right). n = 8 for CTRL-A, CTRL-B, R47H-A, and R47H-B for all baseline conditions; n = 9 for CTRL-A, CTRL-B, R47H-A, and R47H-B for all LPS and IFN-γ treated conditions. *p < .05; ***p < .001. One-way ANOVA with Sidak's test. (c) Volcano plots for genes differentially expressed by LPS treatment in control and TREM2 R47H/+ microglia. (d) The number of differentially expressed genes and enrichment p value (inset) for LPS differentially expressed genes from the “Response to LPS” and “Cell activation” GO terms. (e) Volcano plots for genes differentially expressed by IFN-γ treatment in control and TREM2 R47H/+ microglia. (f) The number of differentially expressed genes and enrichment p value (inset) for IFN-γ- differentially expressed genes from the “Type II interferon” and “Cell activation” GO terms. (g) Heatmap of the gene expression Z-score for selected cytokine and chemokine genes induced by LPS and/or IFN-γ in control and TREM2 R47H/+ microglia. Note that one CTRL-A LPS- treated sample (and thus also a corresponding untreated control) was removed from analysis due to poor read quality.

Article Snippet: Human IL-6 or TNF-α Quantkine ELISA Kits (R + D Systems) were used.

Techniques: Control, Activation Assay, Gene Expression

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Targeting PDXK could promote the formation of TLSs and enhance the efficacy of immunotherapy in gastric cancer. ( A ) Heatmap displaying the metabolic feature for T-cell clusters using scMetabolism package. ( B ) Dot plot showing the expression of the genes encoding rate-limiting enzymes of vitamin B 6 metabolism in different types of T cells. Dot size encodes the percentage of cells expressing the gene, color encodes the average per cell gene expression level. ( C ) Quantification of CXCL13 in PDTFs in the presence of different enzyme inhibitors measured by ELISA. ( D ) Representative H&E staining and PDXK immunohistochemistry of gastric cancer tissues with different responses following immunotherapy. Scale bar, 500 µm. ( E ) The schematic diagram of the animal experiments. ( F–G ) Images of tumors and tumor volume curves of 615 mice treated with various agents (n=6, each group). ( H ) Paraffin sections of mouse subcutaneous graft tumor tissue stained with H&E and IHC detection for CD8, CD20 and CXCL13. Scale bar, 100 µm. ( I ) The number (left panel) and area (right panel) of TLS per tumor area were compared between groups (n=6, each group). Data are presented as the mean±SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. AOX1, aldehyde oxidase 1; CCCP, carbonyl cyanide m-chlorophenyl hydrazone; CR, complete response; MFC, mouse forestomach carcinoma; PBS, phosphate-buffered saline; PDXK, pyridoxal kinase; PDXP, pyridoxal phosphatase; PDTFs, patient-derived tumor fragments; PHOSPHO2, phosphatase orphan 2; PNPO, pyridoxamine 5'-phosphate oxidase; PR, partial response; PSAT1, phosphoserine aminotransferase 1; s.c, subcutaneous injections; SD, stable disease; TLS, tertiary lymphoid structures.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Expressing, Gene Expression, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemistry, Two Tailed Test, Saline, Derivative Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: Intratumoral CXCL13+ CD160+ CD8+ T cells promote the formation of tertiary lymphoid structures to enhance the efficacy of immunotherapy in advanced gastric cancer

doi: 10.1136/jitc-2024-009603

Figure Lengend Snippet: Vitamin B 6 could promote the expression and secretion of CXCL13 in CD160 + CD8 + T cells. ( A ) Quantification of cytokine/chemokine including CXCL13, IL-2, TNF-α and IFN-γ, in PDTFs in the presence of different drugs measured by ELISA. ( B ) The schematic diagram of orthotopic transplanted tumor model with diets containing various amounts of vitamin B 6 (n=6, each group). ( C ) Mouse orthotopic stomach xenograft tumor tissues stained with H&E and IHC detection for CD20 and CXCL13. Scale bar, 500 µm. ( D ) The schematic diagram of orthotopic transplanted tumor model fed with different drugs or diets (n=6, each group). ( E ) The representative images of mouse bioluminescence imaging at week 3 (left panel) and the corresponding quantification analysis (right panel). ( F ) Representative micrographs of xenografts stained with H&E and IHC detection for CD20 and CXCL13. Scale bar, 500 µm. ( G–H ) Density of TLSs (left panel) and ratio of tumor area occupied by TLSs (right panel). ( I ) Gating strategy for CD160 + CD8 + T cells. ( J ) Quantification of cytokine/chemokine including CXCL13, IL-2, TNF-α and IFN-γ, in supernatants from CD160 + CD8 + T-cell cultures in the presence or absence of PL measured by ELISA. ( K ) Flow cytometric analysis and corresponding quantification of CXCL13 + CD160 + CD8 + T cells with or without PL treatment. Data are presented as the mean±SD. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-tailed Student’s t-test. ICIs, immune checkpoint inhibitors; IFN, interferon; IHC, immunohistochemistry; IL, interleukin; MFC, mouse forestomach carcinoma; PBS, phosphate-buffered saline; PL, pyridoxol; PD-1, programmed cell death protein 1; s.c, subcutaneous injections; TLS, tertiary lymphoid structures; TNF, tumor necrosis factor.

Article Snippet: The indicated cytokines and chemokines within the supernatants were detected using human IL-2 ELISA Kit (Solarbio, SEKH-0008), human IFN-γ ELISA Kit (Solarbio, SEKH-0046), human CXCL13 ELISA Kit (Solarbio, SEKH-0072) and human TNF-α ELISA Kit (Solarbio, SEKH-0047) according to the manufacturers’ instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Staining, Imaging, Two Tailed Test, Immunohistochemistry, Saline

Journal: Cancer Research

Article Title: Human 3D Ovarian Cancer Models Reveal Malignant Cell–Intrinsic and –Extrinsic Factors That Influence CAR T-cell Activity

doi: 10.1158/0008-5472.CAN-23-3007

Figure Lengend Snippet: Impaired death receptor signaling in malignant cells caused resistance to CAR T-cell cytotoxicity. A, Hierarchical cluster analysis of transcriptomes for OvCAR3 and G164 cells in monolayer and spheroids. B, Heatmap illustrating normalized gene expression of differentially expressed genes relating to death receptor signaling in OvCAR3 and G164 cells (adjusted P value < 0.05). C, cIAP1/2 expression on human HGSOC omental metastasis ( n = 16) and adjacent omentum ( n = 10). Statistics performed using two-way ANOVA. D and E, Representative images (left) and quantification (right) of Incucyte killing assay in which monolayers of G164 ( D ) and G33 ( E ) cells were treated with birinapant and CAR T cells. F, Western blot showing the expression of cIAP2 in OvCAR3 and G164 cells treated with birinapant. Representative images of three repeats. G, ELISA data showing TNFα concentration after coculturing CAR T cells with monolayers of OvCAR3 and G164 cells for 2 days at 1:5 T:E ratios. Data plotted as mean ± SD for three CAR T-cell donors. Statistics performed using two-way ANOVA. H, Representative images (left) and quantification (right) of Incucyte killing assay in which OvCAR3 monolayer was treated with anti-TNFα antibody and CAR T cells. D–F, Data shown for one CAR T-cell donor at 1:5 T:E ratio. Images shown are 3 days after treatment. Red, dead cells. Scale bars, 400 μm.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) was performed using human IFNα Quantikine kit (R&D Systems, Cat. DIF50C), human TNFα QuantiGlo kit (R&D Systems, Cat. QTA00C), human CCL2/MCP1 Quantikine kit (R&D Systems, Cat. DCP00), and human TGFβ1 Quantikine kit (R&D Systems, Cat. DB100C) according to manufacturer’s instructions.

Techniques: Gene Expression, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Cancer Research

Article Title: Human 3D Ovarian Cancer Models Reveal Malignant Cell–Intrinsic and –Extrinsic Factors That Influence CAR T-cell Activity

doi: 10.1158/0008-5472.CAN-23-3007

Figure Lengend Snippet: Vascularized microfluidic chip to investigate CAR T-cell migration and cytotoxicity. A, Design of the tri-channel microfluidic device with a 2 mm well in the central channel. B, Schematic diagram showing the development of ovarian cancer-on-a-chip model. Scale bar, 600 μm. C, Immunofluorescence image showing microvasculature in fibrin and OvCAR3 collagen gels ( n = 3). Red, HUVEC. Scale bar, 100 μm. D, Real-time images showing the luminal flow of CAR T cells (arrows) through the vasculature formed within the microfluidic device ( n = 2). Green, CAR T cells. Scale bar, 20 μm. E, Immunofluorescence image showing an ovarian cancer-on-a-chip 3 days after CAR T-cell treatment. CAR T cells (arrows) migrated into the OvCAR3 gel in the middle of the device. Dotted line marks the edge of the central well. Green, CAR T cells; red, HUVEC. Scale bar, 100 μm. F and G, CD3 ( F ) and caspase‐3 (Casp3; G ) staining and quantification of OvCAR3 gels isolated from microfluidic device after CAR T-cell and anti-TNFα treatment. H, MSD data showing TNFα, IFNγ, and IL2 concentrations on media from microfluidic devices after CAR T-cell and anti-TNFα treatment. F–H, Data plotted as mean ± SD of two/three gels per two replicates. Two different CAR T-cell donors were used in this experiment. Scale bar, 50 μm. Statistics performed using two-way ANOVA.

Article Snippet: Enzyme-linked immunosorbent assay (ELISA) was performed using human IFNα Quantikine kit (R&D Systems, Cat. DIF50C), human TNFα QuantiGlo kit (R&D Systems, Cat. QTA00C), human CCL2/MCP1 Quantikine kit (R&D Systems, Cat. DCP00), and human TGFβ1 Quantikine kit (R&D Systems, Cat. DB100C) according to manufacturer’s instructions.

Techniques: Migration, Immunofluorescence, Staining, Isolation